Prior to her work in federal politics Ambrose's community service included involvement with organizations working to end violence against women; including the Status of Women Action Group, the Victoria Sexual Assault and Sexual Abuse Crisis Centre, and the Edmonton Women’s Shelter.[1]

Ambrose was first elected as a Member of Parliament in the 2004 federal election.

On February 16, 2005, she made headlines after making a remark in Parliament directed at Liberal Social Development Minister Ken Dryden saying "working women want to make their own choices, we don't need old white guys telling us what to do," in reference to the Liberal national child care plan.

On April 7, 2006, Ambrose announced that Canada had no chance of meeting its targets under the Kyoto Protocol and would have to set more "realistic" goals for cutting greenhouse gases. "My departmental officials and the department officials from natural resources have indicated that it is impossible, impossible for Canada to reach its Kyoto target. And let me be clear. I have been engaging with our international counterparts over the past month, and we are not the only country that is finding itself in this situation." said Ambrose.[4] On June 6, 2006, in a speech she clarified with the following: "So we became the first and only country to publicly state that we know we will not reach our Kyoto targets. This was met by controversy. But we will not be the last. However, no one that follows this debate in any serious manner was surprised to find out that after years of no action on climate change, meeting the onerous targets negotiated under the Liberals was not a reality."

On April 13, 2006, Ambrose was accused of stopping an Environment Canada scientist, Mark Tushingham, from speaking publicly about his own novel. The science fiction novel, entitled Hotter than Hell, posits an alternate future in which global warming has made many parts of the world too hot to live in and has prompted a war between Canada and the U.S. over water resources. Tushingham was scheduled to speak in Ottawa at his book launch about his book and his beliefs underpinning it. However, he claimed an order from Ambrose's office stopped him. In response, Ambrose's spokesperson claimed that the speech was billed as coming from an Environment Canada scientist, who would appear to be speaking in an official capacity, even though his book is a work of fiction.[5]

On May 11, 2006, before the House of Commons, she said "We would have to pull every truck and car off the street, shut down every train and ground every plane to reach the Kyoto target negotiated by the Liberals."[6]

She continued her May 11 speech: "Or we could shut all the lights off in Canada tomorrow -- but that still wouldn’t be enough -- to reach our Kyoto target we’d have to shut off all the lights AND shut down the entire agriculture industry.

"Or instead we could shut down every individual Canadian household, not once, not twice, not three times, but FOUR times over to meet the Kyoto target the Liberals negotiated for Canada.

"Or, we could do what the Liberals thought was the answer faced with the realization that the target they negotiated meant shutting down Canada’s economy -- spend the money overseas buying international credits -- the Liberals had set aside up to $600 per Canadian household to be sent overseas in order to help reach the Kyoto target they negotiated for Canada."

Ambrose was criticized by Bill Graham, Leader of the Opposition, on May 15, 2006 for chairing a UN conference on climate change despite admitting that Canada will not meet its Kyoto Protocol targets to reduce greenhouse gas emissions. Ambrose, who was leading the May 15 to 26 meetings in Bonn, Germany, repeated her claim that Canada's targets under Kyoto are "unachievable." During Question Period that day, Graham asked the prime minister if he saw the "irony" in having someone who "despises" Kyoto to chair the meeting in Germany. At the beginning of the UN conference, Ambrose stated: "I have been very honest with Canadians after the release of our greenhouse gas inventories that we will have great difficulty in meeting those targets. We believe they are unachievable.".[7]

Ambrose told a parliamentary committee that Canada had paid its debts under the Kyoto Protocol only to have an Environment Canada official point out that the bill was still unpaid.[8]

Continued opposition discontent over Ambrose's conduct led the NDP to table a motion in the Commons environmental committee calling for her resignation. The vote, which was held on June 21, 2006, saw the Liberals side with the Conservatives, thus defeating the motion. Had the motion passed, a vote would then have been held in the House of Commons, and, because this was deemed to be a matter of confidence, could possibly have triggered an election.

In August 2006 she stated: “I welcome the commitment from British Columbia to preserve and increase the population of Northern Spotted Owls ... It is my opinion that, given the measures they are taking, such as stopping logging in areas currently occupied by the owls, there is no imminent threat to the survival or recovery of the Northern Spotted Owl at this time.” [9]

On October 19, 2006, Ambrose introduced a Clean Air Act that purported to reduce the level of greenhouse emissions starting in 2020, cutting them to about half of the 2003 levels by 2050. She also introduced other regulations to industries and vehicles as well as a possible cooperation between the federal government and the provinces to create a system that would report air emissions. In an interview with the media, Ambrose denied that the Conservative government had abandoned the Kyoto Protocol despite its previous opposition to it. However, industries will have until 2010 before they are expected to reduce emissions, and the government will not have final (and voluntary) targets ready until 2020. Oil companies will have to reduce emissions on a per-barrel basis, reduction proportional to production basis.[10][11]

News stories began to appear in late 2006 of a possible Cabinet shuffle that included shifting Ambrose from her environment portfolio. On January 4, 2007, Ambrose moved from environment to become Minister of Western Economic Diversification, Minister of Intergovernmental Affairs as well as President of the Queen's Privy Council. The Environment portfolio went to John Baird, the former President of the Treasury Board. On January 19, 2010, Ambrose succeeded Christian Paradis and was named the Minister of Public Works and Government Services. On April 9, 2010 she was also named Minister responsible for the Status of Women after Helena Guergis was dismissed from Cabinet. On July 15, 2013, Ambrose was made Minister of Health and retained the title of Minister of Western Economic Diversification.

On September 26, 2012, Ambrose was in the news again having voted in favour of Motion 312, a motion by Conservative MP Stephen Woodworth that would have directed a Commons committee to revisit the section of the Criminal Code defining at what point human life begins. Viewing the motion as an attempt to re-open debate on abortion laws, Canadian pro-choice groups and Commons opposition parties considered her vote inconsistent with her ministerial role[12][13] and prompted a call for her resignation. The motion was eventually voted down.[14] Ambrose responded to her critics, stating her concern of discrimination against girls that is made possible by sex-selection abortion.[12][13] Pro-Life groups praised Ambrose for supporting the Motion.[15]